Fibroblast-derived exosomes promote epithelial cell proliferation through TGF-ß2 signalling pathway in severe asthma.

BACKGROUND: Bronchial fibroblasts play a key role in airway remodelling in asthma. They regulate epithelial cell functions such as proliferation through growth factors, cytokines, chemokines and exosomes. The role of exosomes in the communication between epithelial cells and fibroblasts by vehiculing these mediators in asthma remains to be determined. OBJECTIVE: To evaluate the role of exosomes released by bronchial fibroblasts on epithelial cell proliferation in severe asthma. METHODS: Exosomes were obtained from culture media of primary bronchial fibroblasts and characterized using Western blot, electron microscopy and flow cytometry. Uptake profile of fluorescent-labelled exosomes in epithelial cells was assessed by flow cytometry. Exosome cytokine content was analysed by Cytokine Arrays. Bronchial epithelial cell proliferation was evaluated by BrdU incorporation test. Exosome biogenesis/release was blocked using sphingomyelinase inhibitor. Plasmid transfection was used to modulate transforming growth factor beta 2 (TGF-ß2) gene expression. RESULTS: We showed that bronchial fibroblasts secreted exosomes, which were internalized by bronchial epithelial cells. Exosomes of severe asthmatic subjects' fibroblasts showed a lower level of TGF-ß2 and significantly increased the epithelial cell proliferation of both healthy and severe asthmatic subjects compared to healthy controls' exosomes. Overexpression of TGF-ß2 in severe asthmatics' fibroblasts induced enhanced TGF-ß2 in exosomes leading to a reduced proliferation of epithelial cells, whereas knockdown of TGF-ß2 enhanced epithelial cell proliferation. CONCLUSION: Our study shows that exosomes are involved in fine-tuning intercellular communication in asthma. Exosomes of severe eosinophilic asthmatics' fibroblasts can contribute to airway remodelling, at least in part, by modulating epithelial cell proliferation observed in severe asthma.

MicroRNA-19a enhances proliferation of bronchial epithelial cells by targeting TGFßR2 gene in severe asthma.

BACKGROUND: Allergic asthma is characterized by inflammation and airway remodeling. Bronchial epithelium is considered a key player in coordinating airway wall remodeling. In mild asthma, the epithelium is damaged and fails to proliferate and to repair, whereas in severe asthma, the epithelium is highly proliferative and thicker. This may be due to different regulatory mechanisms. The purpose of our study was to determine the role of miRNAs in regulating proliferation of bronchial epithelial cells obtained from severe asthmatic subjects in comparison with cells obtained from mild asthmatics and healthy controls. METHODS: Human bronchial epithelial cells (BEC) were isolated by bronchoscopy from bronchial biopsies of healthy donors and patients with mild and severe asthma. MiRNA expression was evaluated using the TaqMan low-density arrays and qRT-PCR. Transfection studies of bronchial epithelial cells were performed to determine the target genes. Cell proliferation was evaluated by BrdU incorporation test. RESULTS: MiR-19a was upregulated in epithelia of severe asthmatic subjects compared with cells from mild asthmatics and healthy controls. Functional studies based on luciferase reporter and Western blot assays suggest that miR-19a enhances cell proliferation of BEC in severe asthma through targeting TGF-ß receptor 2 mRNA. Moreover, repressed expression of miR-19a increased SMAD3 phosphorylation through TGF-ß receptor 2 signaling and abrogated BEC proliferation. CONCLUSION: Our study uncovers a new regulatory pathway involving miR-19a that is critical to the severe phenotype of asthma and indicates that downregulating miR-19a expression could be explored as a potential new therapy to modulate epithelium repair in asthma.

Whole cigarette smoke promotes human gingival epithelial cell apoptosis and inhibits cell repair processes.

BACKGROUND AND OBJECTIVE: Smoking cigarettes increases the risk of developing various types of human diseases, including cancers and periodontitis. As gingival epithelial cells are known to play an active role in innate immunity via the secretion of a wide variety of mediators, and as these cells are the first ones exposed to environmental stimuli such as cigarette smoke, we sought to investigate the effects of whole cigarette smoke on normal human gingival epithelial cells and tissue. MATERIAL AND METHODS: Human gingival epithelial cells were extracted from healthy nonsmokers and used either as a monolayer or as an engineered human oral mucosa to investigate the effect of whole cigarette smoke on cell growth, apoptosis and wound repair/migration. RESULTS: Our findings show that when gingival epithelial cells were exposed once to whole cigarette smoke, this resulted in a significant inhibition of cell growth through an apoptotic pathway, as confirmed by an increase of Bax and a decrease of Bcl-xL and caspase-3 activity. Cigarette smoke also inhibited epithelial cell migration. These effects may explain the disorganization of the engineered human oral mucosa tissue when exposed to whole cigarette smoke. CONCLUSION: Exposure to whole cigarette smoke markedly inhibits epithelial cell growth through an apoptosis/necrosis pathway that involves Bax and Bcl-xL proteins and caspase-3 activity. Cigarette smoke also disrupts epithelial cell migration, which may negatively affect periodontal wound healing.

Monitoring sputum eosinophils in mucosal inflammation and remodelling: a pilot study.

Normalisation of eosinophil counts in sputum of asthmatic patients reduces eosinophilic exacerbations. However, the effect of this strategy on airway remodelling remains to be determined. We compared bronchial inflammation and collagen deposition after 2 yrs of treatment guided by either sputum eosinophils (sputum strategy, SS) or by clinical criteria (clinical strategy, CS). As a pilot study, 20 mild asthmatic patients were randomly assigned to CS or SS strategies. Bronchial biopsies were obtained when minimum treatment needed to maintain control was identified and this was continued for 2 yrs. Biopsies were immunostained for inflammatory cells, mucin 5A (MUC5A) and collagen. The mean dose of inhaled corticosteroids at the start and end of the study was similar in both SS and CS groups. Forced expiratory volume in 1 s increased in both groups at the study end. In SS, mucosal lymphocyte and eosinophil counts, but not neutrophils, were reduced at the end of the study. In CS, only activated eosinophil and neutrophil counts decreased. MUC5A staining decreased in SS but not CS. No change in collagen deposition underneath the basement membrane was observed in either strategy. Treatment strategies that normalise sputum eosinophils also reduce mucosal inflammatory cells and MUC5A expression, but do not change subepithelial collagen deposition in mild to moderate asthma.

Induction of glucocorticoid receptor-beta expression in epithelial cells of asthmatic airways by T-helper type 17 cytokines.

BACKGROUND: Corticosteroid insensitivity in asthmatics is associated with an increased expression of glucocorticoid receptor-beta (GR-beta) in many cell types. T-helper type 17 (Th17) cytokine (IL-17A and F) expressions increase in mild and in difficult-to-treat asthma. We hypothesize that IL-17A and F cytokines alone or in combination, induce the expression of GR-beta in bronchial epithelial cells. OBJECTIVES: To confirm the expression of the GR-beta and IL-17 cytokines in the airways of normal subjects and mild asthmatics and to examine the effect of cytokines IL-17A and F on the expression of GR-beta in bronchial epithelial cells obtained from normal subjects and asthmatic patients. METHODS: The expression of IL-17A and F, GR-alpha and GR-beta was analysed in bronchial biopsies from mild asthmatics and normal subjects by Q-RT-PCR. Immunohistochemistry for IL-17 and GR-beta was performed in bronchial biopsies from normal and asthmatic subjects. The expression of IL-6 in response to IL-17A and F and dexamethasone was determined by Q-RT-PCR using primary airway epithelial cells from normal and asthmatic subjects. RESULTS: We detected significantly higher levels of IL-17A mRNA expression in the bronchial biopsies from mild asthmatics, compared with normal. GR-alpha expression was significantly lower in the biopsies from asthmatics compared with controls. The expression of IL-17F and GR-beta in biopsies from asthmatics was not significantly different from that of controls. Using primary epithelial cells isolated from normal subjects and asthmatics, we found an increased expression of GR-beta in response to IL-17A and F in the cells from asthmatics (P< or =0.05). This effect was only partially significant in the normal cells. Dexamethasone significantly decreased the IL-17-induced IL-6 expression in cells from normal individuals but not in those from asthmatics (P< or =0.05). CONCLUSION: Evidence of an increased GR-beta expression in epithelial cells following IL-17 stimulation suggests a possible role for Th17-associated cytokines in the mechanism of steroid hypo-responsiveness in asthmatic subjects.

Regulation of epithelial cell proliferation by bronchial fibroblasts obtained from mild asthmatic subjects.

BACKGROUND: Bronchial epithelium is considered a key player in coordinating airway wall remodelling. The function of epithelial cells can be modulated by the underlying fibroblasts through autocrine and paracrine mechanisms. OBJECTIVE: To investigate the effect of phenotypic changes in bronchial fibroblasts from asthmatic subjects on epithelial cell proliferation. METHODS: Epithelial cells and fibroblasts derived from bronchial biopsies of asthmatic and healthy controls were cultured in an engineered model. Proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium-bromid (MTT). Epidermal growth factor receptor (EGFR), cyclin-dependent kinase inhibitors p21 and p27 were measured by western blots. Total and active forms of transforming growth factor (TGF)-ß1 were measured using ELISA and bioassay. TGF-ß was inhibited using a recombinant TGF-ß soluble receptor II protein. RESULTS: Proliferation of epithelial cells from asthmatics (AE) is increased when cells were cultured with fibroblasts from normal controls (NF). Fibroblasts from asthmatics (AF) significantly decreased the proliferation of epithelial cells from healthy subjects (NE). Activation of p21, p27, EGFR and TGF-ß1 reflects the proliferation data by decreasing in AE cultured with NF and increasing in NE cultured with AF. Neutralization of TGF-ß increased proliferation of epithelial cells cultured in the asthmatic model. CONCLUSION: Fibroblasts from asthmatic subjects regulate epithelial cell prolifearation, and TGF-ß signalling may represent one of the pathway involved in these interactions.

Developments in the field of allergy in 2008 through the eyes of Clinical & Experimental Allergy.

In 2008, many thousands of articles were published on the subject of allergic disease with over 200 reviews, editorials and original papers in Clinical & Experimental Allergy alone. These represent a considerable amount of data and even the most avid reader could only hope to assimilate a small fraction of this knowledge. There is therefore a pressing need for the key messages that emerge from a journal such as Clinical & Experimental Allergy to be summarized by experts in the field in a form that highlights the significance of the developments and sets them in the context of important findings in the field published in other journals. This also has the advantage of making connections between new data in conditions such as asthma, where articles often appear in different sections of the journal. As can be seen from this review, the body of work is diverse both in terms of the disease of interest and the discipline that has been used to investigate it. However, taken as a whole, we hope that the reader will gain a flavour of where the field is mature, where there remain controversies and where the cutting edge is leading.

Mechanical strain increases cytokine and chemokine production in bronchial fibroblasts from asthmatic patients.

BACKGROUND: Mechanical strain and cytokine stimulation are two important mechanisms leading to airway remodeling in asthma. The effect of mechanical strain on cytokine secretion in airway fibroblasts is not known. The aim of this study was to determine whether bronchial and nasal fibroblasts differentially alter cytokine secretion in response to mechanical strain. METHODS: We measured secretion of the pro-fibrotic cytokine, interleukin-6 (IL-6), and the pro-inflammatory cytokines, IL-8 and monocyte chemotactic protein 1, before and after mechanical strain in bronchial fibroblasts obtained from asthmatic patients [asthmatic bronchial fibroblasts (BAF)] and normal volunteers [normal bronchial fibroblasts (BNF)], and in nasal fibroblasts (NF) obtained from nasal polyps. Cells were grown on flexible membranes and a mechanical strain of 30% amplitude, 1 Hz frequency was applied for 3, 6 and 24 h. Control cells were unstrained. IL-6, IL-8 and monocyte chemotactic protein 1 was measured after 24 h strain using enzyme-linked immunoassay; mRNA was measured by real time polymerase chain reaction. We also measured mRNA for versican, a matrix proteoglycan, known to be upregulated in the asthmatic airway wall. RESULTS: In unstrained conditions, no differences in cytokine secretion were observed. After 24 h strain, BAF secreted more IL-6 than BNF. Mechanical strain increased IL-8 mRNA in BAF, BNF and NF; and IL-6 and versican mRNA, in BAF, only. CONCLUSIONS: Cytokine responses to mechanical strain varied in different airway fibroblast populations, and depended on the site of origin, and the underlying inflammatory state. Strain resulted in IL-6 upregulation and increased message for extracellular matrix protein in bronchial fibroblasts from asthmatic patients only, and may reflect these patients' propensity for airway remodeling.

Role of the pineal gland and melatonin in the photoperiodic control of hypothalamic gonadotropin-releasing hormone in the male jerboa (Jaculus orientalis), a desert rodent.

The neuroendocrine mechanism underlying seasonal changes in gonadal activity of the jerboa, a desert hibernating rodent adapted to harsh climatic conditions, are poorly understood. We investigated the role of the pineal gland and melatonin in the photoperiodic control of hypothalamic gonadotropin-releasing hormone (GnRH). Intact and pinealectomized male jerboas were subjected to short photoperiod, while others were kept under long photoperiod and injected daily with melatonin or vehicle. Testes activity was monitored by evaluating the testes volume during 10 weeks. GnRH immunoreactivity was investigated quantitatively with image analysis. Following melatonin administration, the hormone peaked in plasma after 30 min, with return to control levels 2.5 h later. Exposure to short photoperiod and melatonin resulted in marked increase in the number of GnRH-containing cells in the preoptic area and mediobasal hypothalamus, whereas GnRH immunoreactivity of fibers and terminals in the median eminence decreased under these conditions. The findings indicate that in the jerboa short photoperiod induces testicular regression by prolonging the duration of melatonin as an endocrine signal. This mechanism probably involves inhibition of GnRH release in the median eminence, with consequent accumulation of GnRH in perikarya of the preoptic area and mediobasal hypothalamus. Interestingly, GnRH cells of the median eminence did not appear to be influenced by the photoperiod and pineal melatonin, whereas their number was increased by exogenous melatonin. The latter data suggest for the first time the involvement of an extrapineal melatonin, whose origin remains to be identified, in the modulation of the GnRH regulatory system in rodents.

Eotaxin promotes eosinophil transmigration via the activation of the plasminogen-plasmin system.

The effect of eotaxin, a potent eosinophil chemotactic factor, on eosinophil transmigration through a reconstituted basal membrane (Matrigel) was evaluated. Eotaxin induced significant eosinophil transmigration in the presence of 10% fetal bovine serum (FBS) and interleukin-5. Its effect was optimal at 0.01 microM, and it plateaued at 18 h. Eotaxin's effect was greater with eosinophils from asthmatic subjects (61.1 +/- 3.4%) than with eosinophils from normal subjects (38.7 +/- 4.2%) (P < 0.001). Inhibition of metalloproteinases decreased eotaxin-induced transmigration by < or = 10.4%, whereas inhibition of the plasminogen-plasmin system decreased eotaxin's effect by < or = 44.4% (P = 0.0002). Moreover, eotaxin-induced transmigration was largely diminished in medium with low concentrations of serum [0.5% FBS: 6.1 +/- 2.4%; 10% FBS: 40.2 +/- 5.8% (P = 0.0001)] but returned to its initial level with the addition of plasminogen (2 U/mL) to 0.5% FBS (43.1 +/- 6.5%). These data show that eotaxin is an efficient promoter of eosinophil transmigration in vitro, that it is more potent with cells from asthmatics than with normal cells, and that its effect depends predominantly on the activation of the plasminogen-plasmin system.

Decreased capacity of asthmatic bronchial fibroblasts to degrade collagen.

The mechanisms of fibrillar collagen accumulation in asthmatic bronchi remain unclear, an imbalance between synthesis and degradation of collagen may be implicated in this process. The aim of this study was to compare the capacities of normal (BNF) and asthmatic (BAF) bronchial fibroblasts to degrade collagen. Metalloproteinases and their inhibitors were measured by ELISA, types I and III procollagen synthesis was determined by liquid RIA and, finally, zymography was used to assess the presence of active and latent forms of MMPs. The capacity of fibroblasts to degrade collagen coated onto latex beads was evaluated by flow cytometry. Our results showed that MMP-2 secretion was significantly higher in BNF when compared to BAF and this was confirmed by gelatin zymography. In BNF culture, TIMP-1 and MMP-1 secretions positively correlated with types I and III procollagen synthesis. However, in BAF, this correlation was negative. This suggests that a balance exists between collagen synthesis and degradation in BNF and that this balance is compromised in BAF. On the other hand, BAF did show significantly reduced capacity to degrade collagen when compared to that of BNF. This reduced phagocytic activity was not associated with a decrease in collagen receptor expression. This study establishes for the first time that a relationship exists between metalloproteinases enzyme dysregulation and the reduced capacity of asthmatic bronchial fibroblast to degrade collagen. These events may shed light on why accumulation of collagen can be observed in asthmatic airways.

Effect of luteotrophic hormone (LH) and thymosin on mitogen-induced blast transformation of Balb/c mouse lymphoid cells.

The present study investigates the effect of a pituitary hormone (LH) and a thymic factor (Thymosin Fraction 5 = TF5) on in vitro and in vivo spleen cell proliferative response of 2, 6 and 8-month-old Balb/c mice. In vitro experiments showed that the addition of LH at various concentrations (0.5, 5, 50 or 500 ng/ml) to cultures increased significantly the proliferative response to some known mitogens (ConA, PHA, PWM). The LH stimulation was further enhanced by subsequent addition of TF5 to these cultures. However, the addition of LH to LPS cultures resulted in a blockage of the cell growth which persisted after the addition of TF5. In vivo experiments showed that injection of LH (5 or 50 ng/ml) to 2, 6 and 8-month-old Balb/c mice had a significant increase on blast transformation of lymphoid cells following their incubation with ConA, PHA and PWM, and a significant decrease when they were incubated with LPS. The physiological significance of these findings which point out an intimate connection between immune and endocrine functions is discussed.

Airway inflammation in asthma with incomplete reversibility of airflow obstruction.

This study aimed to determine whether there is a persistent or different type of airway inflammation in patients with an incomplete reversibility of airflow obstruction (IRAO) despite optimal treatment and if so, whether it is associated with an accelerated decline of pulmonary function. Fifteen asthmatic patients with IRAO, and 23 with complete reversibility of airflow obstruction (CRAO) had a spirometry and an induced-sputum (IS) analysis. Past FEV1 values were recorded over 2-12 years during periods of stable asthma. Medians (range) for IS cell differentials were: lymphocytes, 0(0-3)/1(0-2)%; neutrophils, 56(13-88)/38(3-84)% and eosinophils, 2.0(0-82)/4.0(0-68)%, (all P>0.05). Among non-smoking patients, those with IRAO had more neutrophils in IS than those with CRAO (P=0.019). Mean (+/-SEM) yearly fall in FEV1 in IRAO or CRAO patients was 54+/-21/84+/-16 ml/year (P>0.05, predicted age-related decline < or = 26 ml/year, P=0.0008). In the whole group of asthmatic patients, decline of FEV1/year was inversely correlated with the % neutrophils in sputum (r(s)=-0.436, P=0.008) and, in IRAO patients, with the duration of asthma (r(s)=-0.559, P=0.037). In conclusion, persistent airway inflammation and increased decline in pulmonary function can be observed in both asthmatic patients with IRAO/CRAO and are of similar magnitude. Non-smoking patients with IRAO had more neutrophils in IS than CRAO.

Isolation and characterization of human airway fibroblasts in culture.

Asthma is considered an airway inflammatory disorder characterized by variable airflow obstruction and airway hyperresponsiveness (1). The inflammatory component of asthma has been studied extensively over the past few years, but, more recently, the potential contribution of airway wall remodeling to functional and clinical changes has been emphasized (2,3). Although the methods of sampling of bronchial tissue were previously limited, being obtained mostly from autopsic or surgical specimens, they have improved recently.

Airway inflammation and structural changes in airway hyper-responsiveness and asthma: an overview.

Asthma treatment has moved from bronchodilator therapy to an emphasis on anti-inflammatory therapy. Airway inflammation is believed to induce airway hyper-responsiveness (AHR) through the release of mediators that increase the airway response to agonists. However, the exact contribution of airway inflammation in the physiology of airway hyper-responsiveness remains undefined. Structural modifications in airways resulting from inflammation may contribute to the development and persistence of AHR and the development of asthma. This paper reviews some of the main components of airway inflammation and structural changes in asthma, and discusses how these processes may interact to modify airway function and induce respiratory symptoms.

Cysteinyl leukotrienes regulate TGF-ß(1) and collagen production by bronchial fibroblasts obtained from asthmatic subjects.

BACKGROUND: Cysteinyl leukotrienes (CysLTs) play an important role in airway inflammation in asthma but their role in airway remodeling is not completely known. METHODS: CysLTs receptors and procollagen I(α(1)) mRNA were determined by qPCR. Procollagen protein production was measured by RIA and TGF-ß(1) expression was determined by ELISA. TGF-ß receptor expression was assessed by western blots. RESULTS: CysLT1R, TGF-ß-R1 and active TGF-ß(1) are highly expressed in cells from asthmatics compared to normal controls. LTD(4) increased significantly procollagen I(α(1)) mRNA and protein expression in fibroblasts from asthmatics. This increase was blocked by CysLTs receptor antagonist. LTD(4) increased significantly mRNA expression of TGF-ß(1) and active form production in fibroblasts from asthmatics. Inhibition of TGF-ß(1) signaling blocked LTD(4)-induced procollagen I(α(1)) expression. CONCLUSIONS: Fibroblasts from asthmatic subjects express high level of CysLT1R. LTD(4) regulates procollagen I(α(1)) transcription in fibroblasts derived from asthmatic patients by modulating TGF-ß(1) expression. This suggests that CysLTs may play a role in regulating collagen deposition in asthma.